GSM6893692: KO82-35036; Mus musculus; RNA-Seq
An islet RNA was isolated with TRIzol, DNase treated (Turbo DNAse Free; Thermo Fisher Scientific). Samples were rRNA depleted utilizing the RiboGone Mammalian-Low Input Ribosomal RNA Removal Kit (Takara Bio USA Inc., Mountain View, CA, USA), and Agencourt AMPure XP SPRI beads (Beckman Coulter). rRNA depleted total RNA were was assessed for rRNA depletion, RNA integrity, size distribution, and concentration prior to library generation with the Agilent RNA Pico 6000 kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). rRNA depleted RNA passing quality control were was used in the SMARTer Stranded RNA-Seq Kit (Takara Bio USA). Following RNA incubation with stranded N6 primer, buffer, and water, samples were incubated at 94°C for 4 minutes, appropriate for samples with RIN values between 4-7. Following purification of first-strand cDNA, libraries were PCR amplified in accordance with their input RNA amount and purified. Final adapter-ligated cDNA was quantitated by Qubit 3.0 dsDNA high-sensitivity assay kit (Thermo Fisher Scientific) together with qPCR amplification using standard curves of a known concentration of adapter-ligated libraries. Sequencing was performed on the NextSeq 500 with the High Output Reagent Cartridge v2 150 cycles (75bp x 2) (Illumina, San Diego, CA, USA).